WO2009133642A1 - Procédé d'évaluation de l'activité anti-tumorale et procédé de recherche par criblage de substances anti-tumorales par ledit procédé - Google Patents

Procédé d'évaluation de l'activité anti-tumorale et procédé de recherche par criblage de substances anti-tumorales par ledit procédé Download PDF

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WO2009133642A1
WO2009133642A1 PCT/JP2008/070315 JP2008070315W WO2009133642A1 WO 2009133642 A1 WO2009133642 A1 WO 2009133642A1 JP 2008070315 W JP2008070315 W JP 2008070315W WO 2009133642 A1 WO2009133642 A1 WO 2009133642A1
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activity
antitumor
sympathetic nerve
tumor
substance
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PCT/JP2008/070315
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English (en)
Japanese (ja)
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克也 永井
ジアオ シェン
裕子 堀井
芳幸 藤崎
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株式会社Anbas
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a method for evaluating the antitumor action of a test substance.
  • the present invention also relates to a method (screening method) for searching for and obtaining a substance that can be an active ingredient of an antitumor agent using the method.
  • this invention relates to the antitumor agent which uses the substance acquired by the said method as an active ingredient.
  • Cancer is one of the diseases with high morbidity and mortality. Recently, advances in medicine have resulted in an overall decrease in cancer mortality from early detection, but there are no universally effective cancer prevention and treatment methods. Also, due to various recent reasons (increased elderly, environmental pollution, etc.), the number of cancer cases is expected to increase further.
  • cancer treatment is performed by combining one or more aggressive treatments (including surgery, chemotherapy, radiation therapy, or hormone therapy) in addition to early detection.
  • aggressive treatments including surgery, chemotherapy, radiation therapy, or hormone therapy
  • many of these aggressive treatments are invasive and systemic toxicity is unavoidable, so the patient's physical and mental burden is significant, and side effects greatly impair the patient's quality of life. It will be.
  • Some cancer cells or tumor cells are inherently resistant to cytotoxic drugs used in cancer treatment, while others respond initially but remain resistant during treatment.
  • radiotherapy is relatively ineffective at eradicating cancer cells within a solid tumor mass. This is because oxygen-derived free radicals are necessary for cell destruction by radiotherapy (Non-Patent Document 1), but the oxygen level in the tumor mass is low due to the lack of blood supply. Because it is general. It is also known that many chemotherapeutic drugs require oxygen to exert their effectiveness (Non-patent Document 2).
  • cancer The cause of cancer is still unknown. However, it is generally thought that carcinogenesis or carcinogenesis involves a complex process involving multiple genetic and environmental factors. Given the complex interaction of multiple carcinogenic factors, cancer can develop in a specific and universal way to induce cancer cell death or to block tumor growth (cancer cell proliferation). Developing a method is considered a very difficult task.
  • Non-Patent Documents 3 and 4 Recently, methods for enhancing the immune function of a living body through activation of natural killer cells have been proposed for cancer treatment and prevention. It is considered that activating natural killer cells to enhance the immune function of the living body can universally combat cancer regardless of the type of cancer. Gray et al., Brit. J. Radiol. 26: 6: 683 (1953) Giatromanolaki and Harris., Anticancer Res. 21: 4317 (2001) Andre and Anfossi, Methods Mol. Biol. 415: 291-300 (2008) Malmberg et al. Cancer Immunol. Immunother. 2008 Mar 4 [Epubaheat of print] Zea at al. Cancer Res. 65: 3044-3048 (2005)
  • An object of the present invention is to provide a method for evaluating the antitumor action of a test substance. It is another object of the present invention to provide a method for searching for and obtaining a substance that can be an active ingredient of an antitumor agent by using such an evaluation method, that is, a method for screening a new antitumor agent.
  • an object of the present invention is to provide an antitumor agent comprising as an active ingredient a substance having an antitumor action obtained by using the screening method.
  • the present inventors orally administer a mixture of arginine and other amino acids to a test animal transplanted with tumor cells, and determine the size of the tumor (proliferation rate of tumor cells). At the same time, we measured changes in the electrical activity of the centrifugal branch of the sympathetic nerve (splenic sympathetic nerve) that controls the spleen.
  • the present inventor can evaluate the antitumor action of a test substance by using a decrease in splenic sympathetic nerve activity as an index, that is, by using such an evaluation system, It was confirmed that a substance useful as an active ingredient of a tumor agent, or a substance useful as an active ingredient of food or pet food having antitumor activity can be easily searched and found, and the present invention has been completed.
  • the present invention has the following aspects.
  • Method for evaluating the antitumor action of a test substance (I-1) A method for evaluating the antitumor action of a test substance, comprising the following steps: (1) A step of administering a test substance to a non-human animal and measuring splenic sympathetic nerve activity of the non-human animal.
  • (I-2) The method for evaluating antitumor activity according to (I-1), further comprising the following steps: (2) When the activity of the splenic sympathetic nerve of the non-human animal administered with the test substance is lower than the activity of the splenic sympathetic nerve of the non-human animal not administered with the test substance, the test substance has an antitumor effect. The process of judging.
  • (I-3) The method for evaluating antitumor activity according to (I-1) or (I-2), wherein the measurement of splenic sympathetic nerve activity is performed by measuring the electrical activity of the distal branch of the splenic sympathetic nerve.
  • the “method for evaluating the antitumor effect of a test substance” of the present invention can be rephrased as “a method for measuring the antitumor action of a test substance”.
  • Screening method for substance having antitumor action (antitumor substance) (II-1) A method for screening substance having antitumor action from test substances, comprising the following steps: (1) A step of administering a test substance to a non-human animal and measuring the activity of the spleen sympathetic nerve of the non-human animal, and (2) The activity of the splenic sympathetic nerve obtained above is compared with the activity (control) of the spleen sympathetic nerve of a non-human animal not administered with the test substance, and the test substance whose activity is lower than the control is treated with an antitumor A step of selecting as a candidate substance having an action.
  • test substance selected as a candidate substance having an antitumor action in step (2) as an active ingredient of a food, animal feed, or fragrance having an antitumor action, (II-1 ) Or (II-2).
  • Antitumor agent (III-1) A mixture of arginine and lysine, or myrcene, which was evaluated to have antitumor activity by the evaluation method described in any of (I-1) to (I-3) above An antitumor agent containing as an active ingredient.
  • (III-2) The antitumor agent described in (III-1), which is used for treating tumors of human or non-human animals, particularly malignant tumors (cancer).
  • Anti-tumor method An antitumor method in which the antitumor agent described in (III-1) or (III-2) is applied to a human or animal having a tumor.
  • V-1 A mixture of arginine and lysine evaluated as having antitumor activity by the method described in any of (I-1) to (I-3), or myrcene antitumor agent Use as.
  • V-2 For the production of an antitumor agent of arginine and lysine, or myrcene, which is evaluated to have antitumor activity by the method described in any of (I-1) to (I-3) Use of.
  • the presence or absence of the antitumor action of the test substance can be easily determined by administering the test substance and observing the presence or absence of a decrease in the electrical activity of the sympathetic nerve that governs the spleen. it can. That is, according to the present invention, the antitumor action of a test substance can be easily evaluated.
  • the evaluation method of the present invention is useful as a method for evaluating the antitumor action of a conventionally known antitumor agent, particularly an antitumor action based on an immunopotentiating action, and no antitumor action has been known so far.
  • a method for finding a new anti-tumor effect for a new substance including edible substances and fragrance components
  • a method for searching for and acquiring a substance having an anti-tumor action from many substances and It can be effectively used as a method for identifying an active ingredient having an antitumor action from a composition comprising a plurality of ingredients.
  • Newly obtained antitumor substances obtained by such methods include antitumor agents (including human drugs and veterinary drugs), foods, animal feeds, or aromatherapy that have antitumor effects. It can be used effectively as an active ingredient of a fragrance or a fragrance used in the present invention.
  • the method for evaluating antitumor action of the present invention comprises administering a test substance to be evaluated to a non-human animal and measuring the activity of the splenic sympathetic nerve of the non-human animal. Can be implemented.
  • the non-human animal used for the measurement is not particularly limited as long as it is a non-human mammal or a constant temperature animal such as a bird, and examples thereof include rats, mice, guinea pigs, rabbits, monkeys, dogs and cats.
  • Examples of the administration method of the test substance include transdermal administration, oral administration, enteral administration, intravenous administration and nasal administration.
  • Such an administration method can be appropriately selected depending on the purpose of use and the form of use of the test substance.
  • the test substance is a composition for external use (including topical medicine) or its components or is used for the purpose
  • the antitumor action is evaluated by transdermally administering the test substance to a non-human animal. It is preferable.
  • the test substance is an enteral preparation (including those that pass through the intestine, such as oral administration and suppositories) or its components, or if it is used for that purpose, the test substance should be administered to a non-human animal.
  • test substance is an intravenously administered agent (including injections and infusions) or its components, or when it is used for that purpose, the test substance is administered intravenously to a non-human animal, thereby providing an antitumor effect. Is preferably evaluated.
  • test substance is a nasal preparation or a component thereof, or is to be used nasally, such as a fragrance, the test substance is administered to a non-human animal via nasal administration. It is preferable to evaluate the antitumor effect by ingesting the nose.
  • test substance when the test substance is an oral composition (including food, oral medicine, animal feed) or its components, or when it is used for its use, the test substance is administered orally to a non-human animal, It is preferable to evaluate the tumor action. In this case, after the test substance is orally administered, it takes some time until it is absorbed in the body or exerts its effect in the intestine and reflected in the activity of the splenic sympathetic nerve. For this reason, first, the test substance is enterally administered to a non-human animal (enteral small intestine), the antitumor action is evaluated in advance (primary evaluation), and then the test substance whose antitumor action has been confirmed in the primary evaluation is obtained. It can also be administered orally to non-human animals to evaluate the antitumor effect by oral administration (secondary evaluation).
  • Examples of the method of transdermal administration to non-human animals include a method of applying a test substance to the skin of a non-human animal, a method of attaching a sheet impregnated with or carrying a test substance, and the like. Can be appropriately set according to the preparation form (for example, lotion, aerosol, foam, emulsion, cream, ointment, gel, patch, etc.).
  • the site of the non-human animal to be administered transdermally is not limited, but is preferably an area where there is little influence of body hair, specifically a skin area where there is no hair or little hair (eg, abdomen, inside of thigh, tail, etc.) It is.
  • the influence of body hair can be avoided by shaving the test area, the above-mentioned part is not particularly restricted. Moreover, in order to avoid the influence of body hair, an animal having no hair (for example, a hairless mouse, a hairless rat, etc.) can also be used.
  • an animal having no hair for example, a hairless mouse, a hairless rat, etc.
  • a method for intravenous administration to a non-human animal a method of administering a test substance into the vein of a non-human animal using injection or infusion can be used.
  • a test substance is forcibly administered from the nose of the non-human animal using a spray or an inhaler, or in the air of a breeding room
  • a method of spraying a test substance and ingesting the test substance from the nose with breathing can be used.
  • non-human animals are laparotomized under anesthesia, a test substance is administered by placing a cannula in the intestine, or a test substance is administered into the rectum via the anus
  • a method can be mentioned.
  • a method for oral administration to non-human animals a method of forcibly ingesting the test substance by installing a cannula in the oral cavity, or mixing the test animal with food or water and feeding the non-human animal with food or water Can be mentioned.
  • the activity of the sympathetic nerve (spleen sympathetic nerve) that controls the spleen is measured for the non-human animal administered the test substance.
  • the nerve activity can be measured by measuring the electrical activity of the distal branch of the splenic sympathetic nerve.
  • Measurement of electrical activity of splenic sympathetic nerves is specifically performed under anesthesia by detaching a centrifugal branch of splenic sympathetic nerves of a non-human animal under a stereomicroscope and placing it on a silver electrode. This can be done by recording the potential.
  • the silver electrode is preferably sufficiently immersed in a mixture of liquid paraffin and petrolatum in advance.
  • the obtained nerve electrical activity is amplified by a differential amplifier and monitored by an oscilloscope.
  • the noise signal is separated by a window discriminator.
  • the signal is subjected to spike conversion, and the obtained spike is counted as the number of spikes for 5 seconds by a late meter. This is A / D (analog / digital) converted and then recorded on a personal computer (see FIG. 1).
  • the electrical activity of the distal branch of the splenic sympathetic nerve can be measured as the number of spikes. That is, when the number of spikes measured before administration of the test substance is decreased by administration of the test substance, it can be determined that the electrical activity of the splenic sympathetic nerve has been reduced by administration of the test substance.
  • Such a decrease in the electrical activity of the splenic sympathetic nerve correlates accurately with a decrease in the growth rate of the tumor, that is, the antitumor effect, as shown in the experimental examples. For this reason, when the number of spikes measured before administration of the test substance is decreased by administration of the test substance, it can be determined that the electrical activity of the splenic sympathetic nerve has been reduced by administration of the test substance. It can be evaluated that the test substance has an antitumor effect. On the other hand, when the number of spikes measured before administration of the test substance does not vary or increases due to the administration of the test substance, it can be determined that the electrical activity of the splenic sympathetic nerve does not vary or has increased due to the administration of the test substance. In this case, the test substance can be evaluated as having no antitumor action.
  • the antitumor action evaluation method of the present invention can be carried out by performing the following step (1):
  • the method for evaluating an antitumor action of the present invention can be carried out by performing the following step (2) following the above step (1): (2) In the above step (1), the splenic sympathetic nerve activity of the non-human animal administered with the test substance is more than the splenic sympathetic nerve activity (control) of the non-human animal (control non-human animal) not administered with the test substance. A step of determining that the test substance has an antitumor action when the test substance also decreases.
  • the non-human animal used as a control in step (2) may be the same animal as the non-human animal administered the test substance in step (1) or may be a different animal.
  • the activity of the splenic sympathetic nerve measured for the non-human animal before administration of the test substance is used for comparison, and the activity of the splenic sympathetic nerve measured there is used as the control described above.
  • the animal when an animal different from the non-human animal administered with the test substance is used as the control non-human animal, the animal is preferably the same animal as the non-human animal administered with the test substance.
  • the antitumor effect of transdermal administration, intravenous administration, nasal administration, oral administration or enteral administration can be evaluated for the test substance.
  • the test substance to be evaluated for anti-tumor effect can be arbitrarily selected by the measurer.
  • anti-tumor substances and compositions already known for anti-tumor action including pharmaceuticals, foods and fragrances
  • a substance or composition including pharmaceuticals, foods and fragrances
  • the antitumor effect can be reconfirmed by the evaluation method of the present invention, or it can be evaluated that the effect is caused by the activation of natural killer cells (NK cells), and the antitumor effect thereof.
  • NK cells natural killer cells
  • NK cells do not have specificity, it is considered that any tumor can be combated if NK activity is increased. In the latter case, a new antitumor effect can be found for substances and compositions that have not been known to have an antitumor effect until now by the evaluation method of the present invention.
  • the present invention also provides a method for screening a substance having an antitumor action (antitumor substance).
  • the method can be performed by selecting a substance having an action of reducing the activity of the splenic sympathetic nerve of the non-human animal when administered to the non-human animal from many test substances.
  • screening for an antitumor substance can be carried out by performing the following step (1) (i) according to the method for evaluating the antitumor action of the test substance described above.
  • the screening method of the present invention can be carried out by performing the following step (2) following the above step (1): (2) The activity of the splenic sympathetic nerve of the non-human animal administered with the test substance obtained in the above step (1) is compared with the activity (control) of the spleen sympathetic nerve of the non-human animal not administered with the test substance, A step of selecting a test substance whose activity is lower than that of a control as a candidate substance having an antitumor effect.
  • the animal employed in the above-described evaluation method of the present invention can be used in the same manner.
  • the method for measuring the activity of the splenic sympathetic nerve can be performed by measuring the electrical activity of the centrifugal branch of the splenic sympathetic nerve, as in the method employed in the evaluation method of the present invention described above.
  • test substance that is confirmed to have reduced splenic sympathetic nerve activity when administered to a non-human animal can be determined and acquired as a candidate substance (antitumor substance) having antitumor activity. it can.
  • examples of the administration method to non-human animals include transdermal administration, intravenous administration, nasal administration, oral administration and enteral administration, as in the evaluation method of the present invention described above.
  • a test substance that exhibits an antitumor action by skin administration including application and patching
  • enteral administration including small intestine administration and rectal administration
  • test substance that exerts an antitumor action when taken orally it is preferable to administer the test substance orally.
  • a test substance that exerts an antitumor effect by oral first, a test substance that exhibits an antitumor effect by enteral administration is selected in advance by first screening using enteral administration, Subsequently, a secondary screening using oral administration may be performed on the test substance selected here, and a test substance that exhibits an antitumor action by oral administration may be selected.
  • the antitumor substance selected by the screening method of the present invention may be further used for efficacy tests, safety tests, and tests for humans using pathological model animals transplanted with tumor cells. By carrying out these tests, more practical antitumor substances can be selected and obtained.
  • the substances selected in this way are subjected to structural analysis as necessary, and then, depending on the type of the substance, chemical synthesis, biological synthesis (including fermentation), or genetic manipulation, It can be manufactured industrially and can be used as an active ingredient of an antitumor agent, an external composition having an antitumor effect, an oral composition (including food and animal feed), or a fragrance or a fragrance.
  • the present invention also provides an antitumor agent comprising, as an active ingredient, an antitumor substance that is judged to have an antitumor action by the evaluation method of the present invention.
  • the anti-tumor agent includes not only an anti-tumor agent as a human drug but also an anti-tumor agent as a drug for animals (for example, pets such as dogs and cats).
  • Examples of such an antitumor substance include a mixture of arginine and lysine as shown in Experimental Examples 1 and 2. It has been confirmed that the mixture exhibits a decrease in splenic sympathetic nerve activity and a decrease in tumor growth rate (antitumor effect) when administered to a non-human animal at a ratio of 50 mM (preferably orally). ing.
  • the mixture of arginine and lysine can be effectively used as an active ingredient for antitumor, particularly as an antitumor active ingredient that exhibits an antitumor effect by oral administration or enteral administration.
  • a mixture of arginine and lysine can be used as an active ingredient of an antitumor agent having an antitumor effect / efficacy, a food or animal feed, particularly an antitumor agent for oral or enteral administration. That is, according to the present invention, an antitumor agent having an oral or enteral administration form having a mixture of arginine and lysine as an active ingredient and having antitumor efficacy / effect can be provided.
  • the blending amount of arginine and lysine contained in the antitumor agent is not particularly limited as long as the antitumor agent has an antitumor effect, and can be appropriately selected from the range of usually 1 to 100% by weight. Preferably, 50 to 100% by weight can be mentioned.
  • the form of the antitumor agent is not particularly limited as long as it is an oral or enteral administration form. For example, a drink form (drink form), a gel or syrup form, a tablet form, a pill form, a capsule form, a powder or a granular form.
  • enteral dosage forms having general suppository forms such as creams, solids, or capsules.
  • the dose of the mixture of arginine and lysine is not limited, but is preferably 25 mmole or more per dose from the results of experimental examples described later.
  • myrcene can be mentioned as shown in Experimental Examples 3 to 4. It has been confirmed that when the myrcene is ingested nasally by a non-human animal, the splenic sympathetic nerve activity is reduced and the growth rate of the tumor is reduced (antitumor effect).
  • myrcene, especially ⁇ -myrcene (7-methyl-3-methyleneocta-1,6-diene) is contained in plants such as Laurier, Verbena, Caraway, Fennel, Tarragon, Inond, Scarlet Artemisia, Prunus japonica Natural monoterpene hydrocarbon.
  • myrcene can be effectively used as an antitumor active ingredient, particularly as an antitumor active ingredient that exerts an antitumor action when administered nasally or taken nasally. Therefore, myrcene is an antitumor agent that has antitumor effects / efficacy, especially anti-tumor agents for nasal administration, or fragrances that have antitumor effects / efficacy (including aromatherapy agents used for aromatherapy). It can be used as an active ingredient. That is, according to the present invention, it is possible to provide an antitumor agent, a fragrance, and a fragrance that have a nasal administration form that uses myrcene as an active ingredient and has an antitumor effect.
  • the amount of myrcene contained in the antitumor agent and the like is not particularly limited as long as the antitumor agent has an antitumor effect, and can be appropriately selected from the range of usually 1 to 100% by weight.
  • the form of the antitumor agent is not particularly limited as long as it is a nasal administration form. For example, a drop, a spray (a liquid sprayed using a sprayer, an aerosol, etc.), a cleaning agent, an injection, etc. Can do. From the viewpoint of ease of handling, it is preferable to use a push-type container that has a function of releasing a fixed amount of an active ingredient with a single push.
  • the dose of myrcene is not limited, but from the results of experimental examples described later, it should be an amount that evaporates from a 100-fold to 10,000-fold myrcene water dilution suspension at room temperature of 25 ° C and humidity of 60%. Is preferred.
  • the antitumor agent is used by applying once or a plurality of times a day to a human or animal having a tumor, particularly a malignant tumor, for the purpose of antitumor, according to the above form.
  • test sample administration group 2 mM administration group, 10 mM administration group, 50 mM administration group.
  • size of the tumor three times a week is the maximum diameter (the length of the longest part of the tumor, L) and the width of the tumor (W) on the line perpendicular to it is mm. Measured in units and the tumor volume was determined according to the following formula (Gonzalez et al. J. Biol. Chem. 281: 20851-20864 (2006)).
  • mice of all groups (2 mM test sample administration group, 10 mM test sample administration group, 50 mM test sample administration group, water administration control group: 4 mice each), the appearance of the tumor was photographed after the experiment was completed, and then the tumor Was taken out and its weight (g) was measured. All data are shown as mean ⁇ standard error. Statistical tests were performed using analysis of variance (analysis variance, NOVA, with repeated measures) and Mann-Whitney U-test.
  • FIG. 2 shows changes over time in the tumor volume (mm 3 ) of each group (2 mM test sample administration group, 10 mM test sample administration group, 50 mM test sample administration group, water administration control group).
  • formation of a tumor mass exceeding the tumor volume of 400 mm 3 was observed 28 days after human colon cancer cell transplantation (--- ⁇ ---).
  • the tumor volume of the mice in the 10 mM test sample administration group was small until the 18th day after transplantation compared to the tumor volume of the mice in the water administration control group, but rather the mice in the water administration control group by the 28th day. There was no significant difference in the tumor volume between the two groups (--- ⁇ ---).
  • mice in each group were sacrificed by administration of a large amount of urethane anesthetic 28 days after tumor implantation.
  • a photograph of the appearance of the tumor at the time of sacrifice is shown in FIG. 3, and a photograph of the excised tumor mass is shown in FIG. 3 and 4 also show that the tumor mass of the mouse in the 2 mM test sample administration group is larger than the tumor mass of the mouse in the water administration control group, while the tumor mass of the mouse in the 50 mM test sample administration group is large. Was confirmed to be smaller.
  • FIG. 5 shows the weight of the excised tumor mass.
  • the statistical calculation by Mann-Whitney U-test showed no significant difference at a 5% risk rate, but compared to the weight of the tumor mass in the water-administered control group, the tumor mass of the mouse in the 2 mM test sample-administered group On the other hand, the tumor mass weight of mice in the 50 mM test sample administration group tended to decrease (FIG. 5).
  • the tumor increased significantly, and at 10 mM concentration, there was almost no change from the water-treated control group, and at 50 mM concentration, the tumor size decreased significantly.
  • the same tendency was recognized also in the weight of the tumor mass extracted after completion
  • Experimental example 2 (I) Measurement of electrical activity of splenic sympathetic nerve (centrifugal branch) (1) Experimental method (1-1) Preparation of test sample A mixture of arginine and lysine was dissolved in water so as to have respective concentrations of 2 mM, 10 mM, and 50 mM, and this was used as a test sample. Moreover, water was used as a control sample.
  • the test sample prepared above (2 mM, 10 mM, 50 mM Arg + Lys), or only water as a control, 1 ml each was orally administered, and the change in the nerve activity (electric activity) of the distal branch of the splenic sympathetic nerve was measured for 40 minutes immediately after administration.
  • the abdomen was opened under urethane anesthesia in the middle of the light period, and the splenic sympathetic nerve was lifted with a silver electrode, and the method described above (Tanida et al. Am. J. Physiol.
  • changes in splenic sympathetic nerves were measured. Changes in splenic sympathetic nerve activity by oral administration of 1 ml of various test samples (2 mM, 10 mM and 50 mM aqueous solutions) at the time when this measured value settled (around 13:00) was measured electrophysiologically. Specifically, the electrical activity of the splenic sympathetic nerve (centrifugal branch) was measured by lifting the splenic sympathetic nerve branch of the rat onto a silver electrode under a stereomicroscope. In order to prevent drying, the electrode was previously sufficiently immersed in a mixture of liquid paraffin and petrolatum. The obtained nerve electrical activity was amplified with a differential amplifier and monitored with an oscilloscope.
  • the noise signal was separated by a window discriminator and converted into a spike signal.
  • the obtained spike signal was counted as the number of spikes for 5 seconds by a late meter, and was recorded on a personal computer after A / D (analog / digital) conversion (see FIG. 1). Electrical activity was recorded for 40 minutes (see FIG. 6B).
  • the oral administration of the test sample was performed by using a polyethylene tube inserted into the oral cavity and setting the administration rate to 1 ml / min. A tube was inserted into the trachea from the start of the operation to the end of the measurement to secure the airway, and the body temperature (rat rectal temperature) was maintained at 36.0 ⁇ 0.5 ° C. with a heat retaining device. As a control experiment, 1 ml of a control sample (water) was administered into the duodenum under the same conditions.
  • the data of splenic sympathetic nerve activity was analyzed by the average value of the firing frequency (pulse / 5 pulses) per 5 seconds every 5 minutes, and expressed as a percentage with the value before administration as 100%.
  • FIG. 6A shows actual measurement data
  • FIG. 6B is a graph of this actual measurement data, with the actual measurement data before administration (number of spikes) taken as 100%, with an average value every 5 minutes. (0-40 minutes).
  • the top of FIG. 6 (A) is measured data of a rat administered with water as a control (water-treated control rat) [FIG. 6 (B):- ⁇ -], and the second is a 2 mM test sample administered.
  • Measured data of rat (2 mM test sample-administered rat) [FIG. 6B: --- ⁇ -]
  • the splenic sympathetic nerve activity (electrical activity) of water-treated control rats and 10 mM test sample-treated rats was almost the same as that before the administration, The splenic sympathetic nerve activity of the rats treated with 2 mM test sample increased, and the splenic sympathetic nerve activity of the rats treated with 50 mM test sample decreased markedly.
  • the activity of the sympathetic nerve that controls the spleen is controlled in a concentration-dependent manner by the oral administration of the test sample, and the activity of the splenic sympathetic nerve is significantly increased at the concentration of 2 mM, and the splenic sympathetic nerve is increased at the concentration of 50 mM. Activity decreased significantly.
  • Tumor growth rate (%) is 28 days after transplantation in each test sample administration group (2 mM test sample administration group, 10 mM test sample administration group, 50 mM test sample administration group).
  • the tumor volume of the mice was calculated by converting the tumor volume of the mice 28 days after transplantation in the water-administered control group to 100% as a percentage.
  • spleen sympathetic nerve activity inhibition rate (%) was determined based on the graph shown in FIG. Rats, rats treated with 10 mM test sample, rats treated with 50 mM test sample) from 0 to 40 minutes splenic sympathetic nerve activity (Splenic-SNA) -time curve area, spleen from 0 to 40 minutes of water-treated control rats The sympathetic nerve activity value (Splenic-SNA) -calculated by converting the percentage under the time curve to 100%.
  • FIG. 6 (B) is printed on homogeneous paper, and the splenic sympathetic nerve activity value (Splenic-SNA) -time curve from 0 to 40 minutes for each test sample-administered rat and water-administered control rat.
  • the lower area was cut out, the respective weights were measured, and when the weight cut out for the water-administered control rats was taken as 100%, the weight for each test sample-administered rat was converted to a percentage, and the decrease in area was calculated as the inhibition rate.
  • Splenic-SNA splenic sympathetic nerve activity value
  • FIG. 7 shows the results obtained above and graphed with the inhibition rate (%) of splenic sympathetic nerve activity on the horizontal axis and the tumor growth rate (%) on the vertical axis.
  • the numerical formula in the figure of FIG. 7 shows a linear approximation formula and a correlation coefficient.
  • FIG. 7 shows that the suppression rate of splenic sympathetic nerve activity and the growth rate of tumor are in a good correlation, and that the decrease in splenic sympathetic nerve activity reflects the decrease in tumor growth. From this, it is considered that the antitumor wet action of the test substance can be evaluated by measuring the activity (electric activity) of the spleen sympathetic nerve and using the decrease as an index. Moreover, it is thought that the substance which suppresses the activity of a spleen sympathetic nerve suppresses the growth of a tumor by raising NK cells and exhibits an anticancer effect.
  • Experimental example 3 (1) Experimental method (1-1) Preparation of test sample ⁇ -Myrcene (manufactured by Wako Pure Chemical Industries, Ltd.) was suspended in 10,000 times water to prepare a spray solution, which was used as a test sample. Moreover, water was used as a control sample.
  • test animals were bred for 21 days after colon cancer cell transplantation under conditions where the test sample (myrcene diluted suspension) or the control sample (water) was sprayed every day for 30 minutes.
  • the size and weight were measured according to the method described in Experimental Example 1 (Myrcene administration group, Water administration group: 5 mice in each group).
  • Myrcene administration group, Water administration group the appearance of the tumor was photographed after completion of the experiment, and then the tumor was taken out and its weight (g) was measured. All data are shown as mean ⁇ standard error.
  • Statistical tests were performed by analysis of variance (analysis variance, NOVA, with repeated measures) and two-samples t-test.
  • mice in each group were sacrificed 21 days after tumor implantation by administration of a large amount of urethane anesthetic.
  • a photograph of the appearance of the tumor at the time of sacrifice is shown in FIG. 9, and a photograph of the excised tumor mass is shown in FIG. 9 and 10 also confirmed that the tumor mass in the Myrcene administration group was smaller than the size of the tumor mass in the Water administration group.
  • FIG. 11 shows the weight of the extracted tumor mass. It was confirmed that the weight of the tumor mass in the Myrcene administration group was significantly reduced compared to the weight of the tumor mass in the Water administration group (P ⁇ 0.05 ⁇ two-samples t-test).
  • Experimental Example 4 (I) Measurement of electrical activity of splenic sympathetic nerve (centrifugal branch) (1) Experimental method (1-1) Preparation of test sample ⁇ -Myrcene (manufactured by Wako Pure Chemical Industries, Ltd.) was suspended in 100 times water and used as a test sample. Water was used as a control sample.
  • the test sample prepared above Myrcene diluted suspension
  • the control sample water
  • the change in the nerve activity (electric activity) of the distal branch of the splenic sympathetic nerve was measured (three mice in each group).
  • the abdomen was opened under urethane anesthesia in the middle of the light period, and the splenic sympathetic nerve was lifted with a silver electrode, and the method described above (Tanida et al. Am. J. Physiol.
  • the data of splenic sympathetic nerve activity was analyzed by the average value of the firing frequency (pulse / 5 pulses) per 5 seconds every 5 minutes, and expressed as a percentage with the value before administration as 100%.
  • FIG. 12 (A) is actual measurement data, and (B) is a graph of this actual measurement data, with the actual measurement data (number of spikes) before administration taken as 100%, with an average value every 5 minutes. (0-60 minutes).
  • the left column of FIG. 12 (A) is the actual measurement data [FIG. 12 (B): -------] of the rat (Water administration group) which administered water as a control, and the right column administered the test sample.
  • Measured data [Figure 12 (B):- ⁇ -] of rats (Myrcene administration group) is shown.
  • the activity of the splenic sympathetic nerve of the Water administration group was almost the same as that before the administration of water, and the activity of the spleen sympathetic nerve was hardly changed by water stimulation.
  • FIG. 1 It is the schematic which shows the measuring method of the electrical activity of the spleen sympathetic nerve used by this invention.
  • the result of measuring the magnitude of proliferation) over time is shown.
  • Each value represents the average value of 4 mice in each group.
  • (A) is a figure which shows the time-dependent change (actual measurement data) of the activity (electric activity) of the centrifugal branch of a spleen sympathetic nerve when water or a mixture (2 mM, 10 mM, 50 mM) of arginine and lysine is orally administered.
  • (B) is a graph of this (Experimental Example 1).
  • shaft of (B) means the electrical activity of a spleen sympathetic nerve, It graphed by making the electrical activity before oral administration (0 minute) of each test substance into 100%. It is a figure which shows correlation with the suppression rate of a spleen sympathetic nerve activity, and the growth rate (%) of a tumor cell.
  • Tumor formed by HCT116 human colon cancer cell transplantation when water or myrcene (10,000-fold diluted suspension) is ingested nasally into BALB / C mice transplanted with HCT116 human colon cancer cells Shows the result of measurement over time. Each value represents the average value of 5 mice in each group.
  • (A) is a figure which shows the time-dependent change (measurement data) of the activity (electric activity) of the centrifugal branch of the spleen sympathetic nerve when water or myrcene (100-fold diluted suspension) is ingested nasally.
  • (B) is a graph of this (Experimental Example 4).
  • shaft of (B) means the electrical activity of a spleen sympathetic nerve, It graphed by making the electrical activity before ingesting each test substance nasally (0 minute) into 100%.

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Abstract

La présente invention concerne un procédé d'évaluation de l'activité anti-tumorale d'une substance d'intérêt. L'invention concerne également un procédé de recherche par criblage d'une substance candidate (substance anti-tumorale) présentant une activité anti-tumorale, en faisant appel au procédé susmentionné. L'invention concerne encore une substance dont il a été déterminé qu'elle présente une activité anti-tumorale par le procédé d'évaluation susmentionné, substance se révélant donc utilisable en tant qu'agent anti-tumoral. Ce procédé d'évaluation est caractérisé en ce qu'il comporte les étapes suivantes : (1) administration d'une substance d'intérêt à un animal non humain et mesure de l'activité d'un nerf sympathique splénique chez ledit animal non humain ; et éventuellement (2) détermination du fait que la substance présente bien une activité anti-tumorale lorsque l'activité du nerf sympathique splénique chez l'animal non humain ayant reçu la substance baisse par rapport à celle observée chez l'animal non humain n'ayant pas reçu ladite substance.
PCT/JP2008/070315 2008-04-30 2008-11-07 Procédé d'évaluation de l'activité anti-tumorale et procédé de recherche par criblage de substances anti-tumorales par ledit procédé WO2009133642A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005113128A1 (fr) * 2004-05-20 2005-12-01 Eden Research Plc Compositions contenant une particule de glucane creuse ou une particule de paroi cellulaire encapsulant un composant de terpene, procedes de fabrication et d'utilisation associes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005113128A1 (fr) * 2004-05-20 2005-12-01 Eden Research Plc Compositions contenant une particule de glucane creuse ou une particule de paroi cellulaire encapsulant un composant de terpene, procedes de fabrication et d'utilisation associes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NAGAI: "Jiritsu Shinkei Katsudo Sokutei ni Motozuku Kinosei Shokuhin, Flavor no Kaihatsu", FOOD SCIENCE, vol. 49, no. 12, 2007, pages 73 - 80 *
NAGAI: "Jiritsu Shinkei Katsudo Sokutei ni yoru Aroma Seihin, Kenko Shokuhin Oyobi Yakuzai no Kaihatsu Gijutsu [1] I Genrihen Shintai no Kojosei Iji ni Juyo na Yakuwari o Hatasu Jiritsu Shinkei no Katsudo wa Seitai Kino Henka no Shihyo to naru", FOOD & PACKAGING, vol. 47, no. 7, 2006, pages 418 - 426 *
NIIJIMA: "Rat Kyosen Oyobi Hizo o Shihai suru Jiritsu Shinkei no Hataraki ni Oyobosu Arginine- Lycine Jomyakunai Toyo no Koka ni Tsuite", JAPAN SOCIETY FOR THE STUDY OF OBESITY KIROKU, vol. 16TH, 1996, pages 287 - 288 *

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